1. Field of the Invention
The present invention relates generally to the binding of glycoprotein molecules to markers and supports. More particularly, the invention relates to a method for binding immunoglobulins through the carbohydrate moieties on their Fc regions using a hydrazone linkage.
Reagents prepared by binding immunoglobulin (Ig) molecules to a solid phase support matrix are useful in a variety of procedures, such as immunoassays, affinity chromatography, and the like. Generally, such reagents are used to bind and separate substances from a liquid phase, and it is desirable that the immunoglobulin retain as much of its native binding activity as possible. Immunoglobulins, such as IgG, are bivalent, and it is theoretically possible for each mole of bound immunoglobulin to bind up to two moles of its corresponding antigen or hapten. Moreover, immunoglobulins having a desired specificity are expensive to prepare, and it would be desirable to provide methods for their efficient binding to the solid phase without loss and/or inactivation during the binding process.
The most common method for binding immunoglobulins relies on derivatization of the amino groups in the Ig molecule. Although the method is simple and generally successful, the binding efficiency of the resulting product is usually low. Other conventional binding techniques include derivatization of the sulfhydryl groups in the Ig molecule and non-covalently coupling the antibodies through intermediate receptors, such as protein A or anti-F.sub.c antibodies. While such methods are suitable for particular applications, neither provide for efficient utilization of the Ig starting material and binding through the sulfhydryl groups results in a product having a low binding efficiency for the target antigen.
Recently, it has been proposed to bind imununoglobulin molecules to other substances, such as labels and solid phase matrices, through the carbohydrate moieties on their Fc regions. Specifically, aldehydes are formed by selective oxidation of the carbohydrate, and the aldehydes reacted to form either (1) a Schiff base with amino terminal substances or (2) a hydrazone with hydrazide terminal substances. Theoretically, by binding through the Fc region of the Ig molecule, interference with the active Fab regions should be minimized and immunological binding should be maximized. Unfortunately, such binding procedures have not been demonstrated to provide the high binding efficiencies which are theoretically possible. Moreover, the procedures have generally provided for inefficient utilization of the starting Ig material, increasing the cost of the final product.
It would therefore be desirable to provide materials and methods for improving the binding efficiencies of Ig molecules when coupled to solid phase supports. In particular, it would be desirable to provide a method of binding Ig molecules through the carbohydrate groups on their Fc regions to a solid phase with high retention of binding efficiency and efficient utilization of the Ig starting material.
2. Description of the Background Art
Fan and Karush (1983) Fed. Proc. 42:930 (Abstract) briefly describes the binding of IgM to polyacrylic hydrazide agarose. Ito et al. (1986) J. Biochem. 99:1267-1272 describe the binding of disaccharides to epoxy-activated carriers with hydrazine hydrate and adipic acid dihydrazide. Quash et al. (1978) J. Immunol. Meth. 22:165-174 describe the binding of various substances, including IgG, to latex particles substituted with hydrazine groups. Aldehyde groups on the IgG were generated with sodium periodate and the aldehydes and hydrazine groups reacted to form hydrazone linkages. Chua et al. (1984) Biochem. Biophys. Acta 800:291-300 describes the binding of IgM to liposomes through hydrophobic hydrazides. O'Shannessy et al. (1984) Immunol. Lett. 8:273-277 describes the biotinylation of immunoglobulins through aldehydes provided on the carbohydrate moiety of the immunoglobulin and subsequent reaction with biotin hydrazide. European patent application No. 88,695 describes methods for binding immunoglobulins to a solid phase, such as an amine agarose, through aldehydes on the carbohydrate moieties of the immunoglobulin. The application also suggests the use of hydrazides for coupling carbohydrates.